supplemental information underwood et al. an arabidopsis lipid … · 2017. 6. 6. · pen1 signal...

Supplemental Information

Underwood et al.

An Arabidopsis lipid flippase is required for timely recruitment of defenses to the host-

pathogen interface at the plant cell surface.

Supplemental Figure 1. Localization of PEN3-GFP in twenty Arabidopsis mutants

displaying aberrant localization of PEN3 (alp). Z-projected confocal micrographs showing

PEN3-GFP localization in the parental line and mutants alp1-alp20 are presented. Mutants are

grouped according to phenotypic categories indicated by numbers in the upper right corner of

images. 1 – ER localization; 2 – punctate localization; 3 – reduced focal accumulation at

penetration sites; 4 – focal accumulation in the absence of pathogen stimulus. Images were

collected at 24h after inoculation of leaves with Bgh. Arrows indicate sites of attempted

penetration by Bgh. Arrowheads (alp9, alp14, alp18) indicate spontaneous focal accumulation of

PEN3-GFP in epidermal cells of uninoculated leaves. Scale bars = 20 μm.

Supplemental Figure 2. Quantification of GFP-labeled endomembrane compartments in mutant backgrounds and after inhibitor treatments. (A) Numbers of endomembrane compartments (EC) positive for PEN3-GFP signal in parental line, alp3, and PEN3-GFP/ala3-6 leaf tissue. * indicates significant difference compared to PEN3-GFP according to Tukey’s post-hoc test (P < 0.01). (B) Numbers of EC positive for PEN3-GFP signal in alp3 leaf tissue at 30 min (left) or 60 min (right) after treatment with 75 μg/ml wortmannin, 100 μM tyrphostin A23, 100 μM tyrphostin A51, or mock treatment. * indicates significant difference compared to mock treatment according to Tukey’s post-hoc test (P < 0.01). (C) Numbers of EC positive for GFP-PEN1 signal in GFP-PEN1 or GFP-PEN1/ala3-6 leaf tissue. * indicates a significant difference (P < 0.01; Student’s T-test). For all panels punctate EC were enumerated for 5 images per line/treatment using the particle counting function of ImageJ software (see Methods for details). Average numbers of EC carrying PEN3-GFP or GFP-PEN1 are plotted and error bars represent SD (n = 5).

Supplemental Figure 3. The alp3 mutant exhibits morphological defects associated with mutation in ALA3. (A) Rosette diameters (mm) of Col-0, parental line, pen3-3, alp3, ala3 T-DNA mutants, and F1 individuals from an alp3 x ala3-6 cross. Rosettes of five 3-week-old plants were measured for each line. Error bars indicate SD (n=5). Asterisks indicate significant differences from Col-0 determined using Tukey’s post-hoc test (P < 0.01). (B) Root morphologies of Col-0, parental line, pen3-3, alp3, ala3 T-DNA mutants, and F1 individuals from an alp3 x ala3-6 cross. Representative 7-day-old seedlings are shown for each line. (C) Root lengths (mm) of Col-0, parental line, pen3-3, alp3, ala3 T-DNA mutants, and F1 individuals from an alp3 x ala3-6 cross. Roots of ten 7-day-old plants were measured for each line. Error bars indicate SD (n=10). Asterisks indicate significant differences from Col-0 determined using Tukey’s post-hoc test (P < 0.01).

Supplemental Figure 4. ALA3 transcript accumulation in alp3 and ala3-6 mutants. (A) Relative ALA3 mRNA abundance in parental line, alp3, and ala3-6 leaf tissue determined by qPCR. (B) rtPCR amplification products of the first five exons of ALA3 mRNA from the parental line, ala3-6, and alp3 illustrating a transcript splicing defect in the alp3 mutant. Amplification products for the first five exons of ALA3 are shown in the upper panel, ACT2 control is shown in the lower panel.

Supplemental Figure 5. Transfected TGN and Golgi markers show expected localizations in protoplasts in control experiments. Image panels show single confocal optical sections collected from protoplasts derived from VTI12-GFP transgenic plants transfected with a plasmid carrying VTI12-mCHERRY (A) or protoplasts derived from MEMB12-GFP transgenic plants transfected with a plasmid carrying G-rb (B). GFP signal is shown in green in the left panels, mCHERRY signal is shown in magenta in the middle panels, and merged images, with white indicating co-localization, are shown in the right panels. Scale bars = 10 μm.

Supplemental Figure 6. Brefeldin A (BFA) treatment of alp3 mutant leaves causes aggregation of endomembrane compartments carrying PEN3-GFP. Z-projected confocal micrographs illustrating localization of PEN3-GFP in the alp3 mutant background (upper panels) or the TGN marker VTI12-YFP in wt background (lower panels) after treatment with 100 μg/ml BFA (right panels) or a mock, solvent only control (left panels). Images were collected at 3 h after syringe infiltration of leaves from 3-week-old plants. Scale bars = 20 μm.

Supplemental Figure 7. Effect of wortmannin and tyrphostins on PEN3-GFP endomembrane accumulation in alp3 at 30 min. Z-projected confocal micrographs illustrating PEN3-GFP localization in 3-week-old alp3 leaves 30 min after treatment with 75 μg/ml wortmannin, 100 μM tyrphostin A23, 100 μM tyrphostin A51, or mock treatment. Scale bars = 20 μm.

Supplemental Figure 8. mCHERRY-PEN1 shows expected PM localization when transfected into parental line protoplasts. Image panels show single confocal optical sections collected from protoplasts derived from the parental line transfected with a plasmid carrying mCHERRY-PEN1. (A) PEN3-GFP signal is shown in green in the left panel, mCHERRY-PEN1 signal is shown in magenta in the middle panel, and merged images, with white indicating co-localization, are shown in the right panel. Scale bars = 10 μm.

Supplemental Figure 9. Recruitment of GFP-PEN1 and deposition of callose at sites of pathogen detection are delayed in the ala3 mutant. (A) Timecourse evaluation of frequencies of flg22-induced GFP-PEN1 focal accumulation (FA) in wt (white bars) or ala3-6 mutant (gray bars) backgrounds. Leaves were treated with 5 μm flg22 and GFP-PEN1 FAs were enumerated for 20 random microscope fields of view per leaf for 3 leaves per line at 6 h, 9 h, 12 h, and 24 h after flg22 treatment. (B) Frequencies at which GFP-PEN1 was observed at the tips of Bgh appressoria at 12 hpi and 24 hpi in wt (white bars) and ala3-6 mutant (gray bars) backgrounds. Accumulation of GFP-PEN1 was scored at the tips of 25 Bgh appressoria per leaf for 3 leaves per time-point for each line. (C) Frequencies at which callose was observed at the tips of Bgh appressoria at 12 hpi and 24 hpi in wt (white bars) and ala3-6 mutant (gray bars) backgrounds. Accumulation of aniline blue-stained callose deposits was scored at the tips of 50 Bgh appressoria per leaf for 3 leaves per time-point for each line. In all panels, error bars represent SD (n=3) and asterisks indicate significant differences compared to wt (P < 0.01; Student’s T-test).

Supplemental Figure 10. PAMP treatment causes increased accumulation of PEN3-GFP and GFP-PEN1 in endomembrane compartments. (A) Intensity of PEN3-GFP signal in endomembrane compartments (EC) compared to background at 4 h after 5 μM flg22, 100 μg/ml chitin, or mock treatment. Pixel intensities were determined for 10 spots representing apparent EC or 10 spots representing background signal for mock (white bars), flg22 (gray bars), and chitin (black bars) treatments using ImageJ software. Average intensities are plotted. (B) Intensity of GFP-PEN1 signal in EC compared to background at 4 h after 5 μM flg22, 100 μg/ml chitin, or mock treatment. Pixel intensities were determined for 10 spots representing apparent EC or 10 spots representing background signal for mock (white bars), flg22 (gray bars), and chitin (black bars) treatments using ImageJ software. Average intensities are plotted. In panels A and B, error bars represent SD (n=10) and means indicated by the same letter are not significantly different according to Tukey’s post-hoc test (P < 0.01). (C) Numbers of EC positive for PEN3-GFP or GFP-PEN1 signal at 4 h after 5 μM flg22, 100 μg/ml chitin, or mock treatment. Punctate EC were enumerated for 5 images per line for each treatment using the particle counting function of ImageJ software (see Methods for details). Average numbers of EC carrying PEN3-GFP or GFP-PEN1 detected after mock (white bars), flg22 (gray bars), or chitin (black bars) treatment are plotted. Error bars represent SD (n=5) and means indicated by the same letter are not significantly different according to Tukey’s post-hoc test (P < 0.01).

Supplemental Figure 11. Relative transcript abundance of flg22 responsive genes in ala3 mutants compared to wild-type. (A) Relative FRK1 (left) or At2g17740 (right) mRNA abundance in untreated leaf tissue of Col-0, ala3-6, the parental line, and alp3, determined by qPCR. (B) Relative FRK1 mRNA abundance in H2O (left) or flg22 (right) treated leaf tissue of Col-0, ala3-6, the parental line, and alp3 at 12h (grey bars) and 24h (black bars) after treatment determined by qPCR. (C) Relative At2g17740 mRNA abundance in H2O (left) or flg22 (right) treated leaf tissue of Col-0, ala3-6, the parental line, and alp3 at 12h (grey bars) and 24h (black bars) after treatment determined by qPCR.

Supplemental Table 1. Co-localization analyses for PEN3-GFP and endomembrane markers in alp3 protoplasts or control transfections of markers for the same compartment in wt protoplasts. For each analysis, co-localization between GFP and mCHERRY is displayed as the mean of the percentages of overlapping fluorescent signals in 30 manually selected regions of interest from 15 different transfected protoplasts and as the mean of Pearson correlation coefficients derived from the same set of 30 images. Error is displayed as SD. * indicates a P value < .0001 for the comparison of values for actual co-localization to values for co-localization of randomly distributed particles by Student’s T-test.

Co-localization between PEN3-GFP and VTI12-mCH in alp3 protoplasts

Actual co-localization Co-localization of randomly distributed

particles

% PEN3-GFP w/

VTI12-mCH

% VTI12-mCH w/

PEN3-GFP

Pearson Correlation Coefficient

% PEN3-GFP w/

VTI12-mCH

% VTI12-mCH w/

PEN3-GFP


76 ± 13* 85 ± 10* 0.84 ± 0.10* 15 ± 13 18 ± 16 -0.12 ± 0.30

Co-localization between PEN3-GFP and mCH-SYP61 in alp3 protoplasts


particles

% PEN3-GFP w/

mCH-SYP61

% mCH-SYP61 w/

PEN3-GFP


% PEN3-GFP w/ mCH-SYP61

% mCH-SYP61 w/

PEN3-GFP


74 ± 15* 76 ± 13* 0.80 ± 0.13* 18 ± 16 17 ± 16 -0.10 ± 0.31

Co-localization between PEN3-GFP and G-rb in alp3 protoplasts


particles

% PEN3-GFP w/ G-rb

% G-rb w/ PEN3-GFP


% PEN3-GFP w/ G-rb

% G-rb w/ PEN3-GFP


38 ± 21* 25 ± 17* 0.12 ± 0.20* 12 ± 10 11 ± 10 -0.26 ± 0.23

Co-localization between VTI12-GFP and VTI12-mCH in wt protoplasts


particles

% VTI12-GFP w/

VTI12-mCH

% VTI12-mCH w/

VTI12-GFP


% VTI12-GFP w/

VTI12-mCH

% VTI12-mCH w/

VTI12-GFP


76 ± 10* 77 ± 15* 0.72 ± 0.14* 19 ± 15 20 ± 17 -0.24 ± 0.22

Co-localization between MEMB12-GFP and G-rb in wt protoplasts


particles

% MEMB12-GFP w/ G-rb

% G-rb w/ MEMB12-

GFP


% MEMB12-GFP w/ G-rb

% G-rb w/ MEMB12-

GFP


86 ± 7* 87 ± 10* 0.80 ± 0.10* 21 ± 19 21 ± 18 -0.07 ± 0.25

Supplemental Table 2. Co-localization analysis for PEN3-GFP and FM4-64 in alp3 protoplasts. Co-localization between GFP and FM4-64 is displayed as the mean of the percentages of overlapping fluorescent signals in 30 manually selected regions of interest from 15 different transfected protoplasts and as the mean of Pearson correlation coefficients derived from the same set of 30 images. Error is displayed as SD. * indicates a P value < .0001 for the comparison of values for actual co-localization to values for co-localization of randomly distributed particles by Student’s T-test.

Co-localization between PEN3-GFP and FM4-64 in alp3 protoplasts


particles

% PEN3-GFP w/ FM4-64

% FM4-64 w/ PEN3-

GFP


% PEN3-GFP w/ FM4-64

% FM4-64 w/ PEN3-

GFP


71 ± 13* 82 ± 10* 0.77 ± 0.11* 11 ± 12 13 ± 16 -0.30 ± 0.28

Supplemental Table 3. Co-localization analysis for PEN3-GFP and mCHERRY-PEN1 in alp3 protoplasts. Co-localization between GFP and mCHERRY displayed as the mean of the percentages of overlapping fluorescent signals in 30 manually selected regions of interest from 15 different transfected protoplasts and as the mean of Pearson correlation coefficients derived from the same set of 30 images. Error is displayed as SD. * indicates a P value < .0001 for the comparison of values for actual co-localization to values for co-localization of randomly distributed particles by Student’s T-test.

Co-localization between PEN3-GFP and mCH-PEN1 in alp3 protoplasts


particles

% PEN3-GFP w/

mCH-PEN1

% mCH-PEN1 w/

PEN3-GFP


% PEN3-GFP w/

mCH-PEN1

% mCH-PEN1 w/

PEN3-GFP


68 ± 16* 69 ± 16* 0.64 ± 0.13* 18 ± 13 15 ± 11 -0.28 ± 0.22

supplemental information underwood et al. an arabidopsis lipid … · 2017. 6. 6. · pen1 signal...

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